Dual markers

Abstract

The invention is directed to methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are misaminoacylated with non-radioactive markers which may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. Markers may comprise cleavable moieties, detectable labels, reporter properties wherein markers incorporated into protein can be distinguished from unincorporated markers, or coupling agents which facilitate the detection and isolation of nascent protein from other components of the translation system. The invention also comprises proteins prepared using misarminoacylated tRNAs which can be utilized in pharmaceutical compositions for the treatment of diseases and disorders in humans and other mammals, and kits which may be used for the detection of diseases and disorders.

What is claimed is:  
     
       1. A method for incorporating a marker into a protein, comprising: 
       a) providing a misaminoacylated initiator tRNA molecule which only recognizes the first AUG codon that serves to initiate protein synthesis, said misaminoacylated initiator tRNA molecule comprising a fluorescent marker; and  
       b) introducing said misaminoacylated initiator tRNA to a translation system under conditions such that said fluorescent marker is incorporated into a nascent protein.  
       wherein said nascent protein, after step b) is complete, comprises a fluorescent marker and an affinity marker.  
     
     
       2. The method of  claim 1 , further comprising, after step b), isolating said nascent protein. 
     
     
       3. The method of  claim 1 , wherein said translation system comprises a cell-free translation system. 
     
     
       4. The method of  claim 3 , wherein said cell-free translation system is selected from wheat germ extracs,  E. Coli  cell lysates, and rabbit reticulocyte lysates. 
     
     
       5. The method of  claim 1 , wherein, after step b), said fluorescent marker is detected. 
     
     
       6. A method for incorporating a marker into a protein, comprising: 
       a) providing: i) a sample suspected of containing nucleic acid having one or more chain truncating mutations, and ii) a misaminoacylated tRNA molecule, said misaminoacylated tRNA molecule comprises a first marker; and  
       b) introducing said nucleic acid sample and said misaminoacylated tRNA into a translation system under conditions such that said first marker is incorporated into a nascent protein.  
     
     
       7. The method of  claim 6 , wherein said sample contains a chain truncating mutation and said nascent protein is truncated. 
     
     
       8. The method of  claim 6 , wherein said chain truncating mutation comprises a frameshift mutation. 
     
     
       9. The method of  claim 6 , wherein said first marker comprises a detectable label. 
     
     
       10. The method of  claim 9 , wherein said label has a detectable electromagnetic spectral property. 
     
     
       11. The method of  claim 9 , wherein said label comprises a fluorescent moiety. 
     
     
       12. The method of  claim 6 , wherein said nascent protein, after step b) is complete, comprises first and second markers. 
     
     
       13. The method of  claim 12 , wherein said second marker comprises a detectable marker. 
     
     
       14. The method of  claim 12 , wherein said second marker comprises an affinity marker. 
     
     
       15. The method of  claim 6 , wherein said first marker comprises an affinity marker. 
     
     
       16. The method of  claim 6 , further comprising, after step b), isolating said nascent protein. 
     
     
       17. The method of  claim 6 , wherein said translation system comprises a cell-free translation system. 
     
     
       18. The method of  claim 17 , wherein said cell-free translation system is selected from wheat germ extracs,  E. coli  cell lysates, and rabbit reticulocyte lysates. 
     
     
       19. The method of  claim 6 , wherein, after step b), said first marker is detected. 
     
     
       20. The method of  claim 6 , wherein said misaminoacylated tRNA is an initiator tRNA. 
     
     
       21. The method of  claim 6 , wherein said misaminoacylated tRNA is a suppressor tRNA. 
     
     
       22. A method for incorporating a marker into a protein, comprising: 
       a) providing: i) a sample suspected of containing nucleic acid having one or more frameshift mutations, and ii) a misaminoacylated tRNA molecule, said misaminoacylated tRNA molecule comprises a first marker; and  
       b) introducing said nucleic acid sample and said misaminoacylated tRNA into a translation system under conditions such that said first marker is incorporated into a nascent protein.  
     
     
       23. The method of  claim 22 , wherein said transcript comprises a frameshift mutation which generates a stop codon resulting in chain termination. 
     
     
       24. The method of  claim 22 , wherein said transcript comprises a frameshift mutation and said nascent protein comprises an amino acid sequence that is altered. 
     
     
       25. The method of  claim 22 , wherein said first marker comprises a detectable label. 
     
     
       26. The method of  claim 25 , wherein said label has a detectable electromagnetic spectral property. 
     
     
       27. The method of  claim 25 , wherein said label comprises a fluorescent moiety. 
     
     
       28. The method of  claim 22 , wherein said nascent protein, after step b) is complete, comprises first and second markers. 
     
     
       29. The method of  claim 28 , wherein said second marker comprises a detectable marker. 
     
     
       30. The method of  claim 28 , wherein said second marker comprises an affinity marker. 
     
     
       31. The method of  claim 22 , wherein said first marker comprises an affinity marker. 
     
     
       32. The method of  claim 22 , further comprising, after step b), isolating said nascent protein. 
     
     
       33. The method of  claim 22 , wherein said translation system comprises a cell-free translation system. 
     
     
       34. The method of  claim 33 , wherein said cell-free translation system is selected from wheat germ extracs,  E. coli  cell lysates, and rabbit reticulocyte lysates. 
     
     
       35. The method of  claim 22 , wherein, after step b), said first marker is detected. 
     
     
       36. The method of  claim 22 , wherein said misaminoacylated tRNA is an initiator tRNA. 
     
     
       37. The method of  claim 22 , wherein said misaminoacylated tRNA is a suppressor tRNA. 
     
     
       38. A method for incorporating a marker into a protein, comprising: 
       a) providing a misaminoacylated tRNA molecule which only recognizes the first codon designed to serve to initiate protein synthesis, said misaminoacylated tRNA molecule comprising a fluorescent marker; and  
       b) introducing said misaminoacylated tRNA to a translation system under conditions such that said fluorescent marker is incorporated into a nascent protein,  
       wherein said nascent protein, after step) b) is complete comprises a fluorescent marker and an affinity marker.  
     
     
       39. The method of  claim 38 , further comprising, after step b), isolating said nascent protein. 
     
     
       40. The method of  claim 38 , wherein said translation system comprises a cell-free translation system. 
     
     
       41. The method of  claim 40 , wherein said cell-free translation system is selected from wheat germ extracs,  E. coli  cell lysates, and rabbit reticulocyte lysates. 
     
     
       42. The method of  claim 38 , wherein, after step b), said fluorescent marker is detected. 
     
     
       43. The method of  claim 38 , wherein said misaminoacylated tRNA is an initiator tRNA. 
     
     
       44. The method of  claim 38 , wherein said misaminoacylated tRNA is a suppressor tRNA.